System and method for macroscopic and microscopic imaging ex-vivo tissue

ABSTRACT

A system having a macroscopic imager, a microscopic imager, and a stage for moving a substrate supporting ex-vivo tissue with respect to each of the imagers to enable the macroscopic imager to capture macroscopic images, and the microscopic imager to capture optically formed sectional microscopic images on or within the tissue, when presented to the tissue, via the optically transparent material of the substrate. A computer system controls movement of the stage, and receives the macroscopic and microscopic images. A display is provided for displaying the macroscopic and microscopic images when received by the computer system. The tissue is verified as being in an orientation at least substantially flush against the upper surface of the substrate by being in focus in displayed macroscopic images prior to imaging by the microscopic imager, and if needed, any portion of the tissue unfocused is manually positioned until desired tissue orientation is achieved.

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/635,530, filed Feb. 26, 2018, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to a system and method for macroscopic andmicroscopic imaging ex-vivo tissue, and particularly to, a system havingmacroscopic and microscopic imagers mounted in a common housing forimaging an ex-vivo tissue sample with a movable stage to present thetissue sample to each of the imagers, and a method by which an ex-vivotissue sample is presented to such imagers to carry out imaging of theex-vivo tissue sample. The tissue sample is supported on a substratepresented by the stage to each of the macroscopic and microscopicimagers for capturing images of the tissue sample via opticallytransparent material of the substrate. As it is important that areas ofinterest of the ex-vivo tissue sample to be imaged by the microscopicimager lie flat or substantially flush against the substrate whichsupports the tissue sample, the present invention is particularly usefulto verify that the tissue sample lies in such orientation usingdisplayed images from the macroscopic imager prior to imaging by themicroscopic imager, and if needed, manual reorienting any portion of thetissue sample with respect to the substrate until desired tissue sampleorientation is achieved. While the microscopic imager is preferably aconfocal microscope providing optically sectioned microscopic images ofthe tissue, and at different depths within the tissue, other microscopicimagers enabling optically sectioned images of tissue may also be used.

BACKGROUND OF THE INVENTION

In Mohs micrographic surgery, tissue having a tumor, typically acarcinoma on the skin of the head or neck, is excised from a patientunder microscopic guidance. The excised tissue specimen, often called abiopsy, is horizontally sliced to provide thin tissue sections that arethen histologically prepared on slides (i.e., slicing, slide mounting,and staining). The slides are reviewed under a microscope to determinewhether the tumor is fully contained in the excised tissue. This isindicated by the absence of the tumor in the edges or margins of theexcised tissue. If the tumor is not fully contained in the excisedtissue, additional tissue from the patient is excised and the procedureis repeated until all tissue sections taken indicate the tumor has beenremoved from the patient. Mohs surgery permits removal of a tumor withmaximum preservation of normal surrounding tissue.

To prepare each ex-vivo tissue specimen removed by the patient duringMohs surgery, multiple sections or slices are manually made with amicrotome, where each section is planar and parallel to each other.Often the tissue specimen is first frozen to make the tissue easier tomanipulate and cut by the microtome. However, since numerous sectionsmust be made from each tissue specimen and then histologically preparedon slides, this procedure is both tedious and time consuming.

Confocal microscopes optically section tissue to produce microscopicimages of tissue sections without requiring such histologicalpreparation of the tissue on slides. An example of a confocal microscopeis the VivaScope® manufactured by Caliber Imaging Diagnostics, Inc. ofRochester, N.Y., U.S.A. Other examples of confocal microscopes aredescribed in U.S. Pat. Nos. 5,788,639, 5,880,880, 7,394,592, and9,055,867. Optically sectioned microscopic images of tissue can also beproduced by optical coherence tomography or interferometry, such asdescribed in Schmitt et al., “Optical characterization of diseasetissues using low-coherence interferometry,” Proc. of SPIE, Volume 1889(1993), or by a two-photon laser microscope, such as described in U.S.Pat. No. 5,034,613.

One problem with optical sectioning an ex-vivo tissue sample is thattissue often does not lie flat upon a substrate of optically transparentmaterial, e.g., glass or plastic, through which images of the tissuespecimen can be captured, such as due to folding of the tissue, or airbubbles trapped between the tissue and substrate. This problem isexacerbated when the tissue sample is thick, such as 2-3 mm, such thatside edges containing possible tissue margins do not lie flat upon thesubstrate for microscopic imaging. To overcome this problem, a cassetteis described in U.S. Pat. No. 6,411,434 having a base member with arigid optically transparent planar window upon which a tissue specimenis situated, and a pliable plastic membrane locatable over the windowand a substantial portion of the base member through which the tissuespecimen can be manually reoriented. Although useful, it needs a skilledtechnician using a probe to reorient the tissue under the membrane to adesired position without puncturing the membrane. This is a delicateprocedure which if not performed properly can damage the tissuespecimen. Moreover, it has been found that despite training, technicianshave had difficulty determining where to manipulate tissue, and caninadvertently damage tissue by applying more pressure with the probethan needed, or manipulate tissue already sufficiently planar againstthe substrate for optical sectioning.

An additional problem is that even once a specimen visually appears to atrained technician to be properly oriented, one cannot readily verifythat the tissue specimen is properly oriented against the substrate, orif manipulated that such manipulation succeeded, so that the portions ofinterest of the tissue can be fully presented to the confocal microscopeby being substantially flush against the substrate through which imagingis carried out. This is important so that margins along the tissue arenot missed when determining healthy from abnormal tissue (e.g., cells)associated with the tumor (or a lesion) being removed. While U.S. Pat.No. 6,411,434 further describes a camera for capturing a macroscopicimage of a tissue specimen once oriented in a cassette, such camera isnot positioned to image the tissue via the same substrate as theconfocal imager that images the tissue. Thus, it would be desirable toboth verify that the ex-vivo tissue is sufficiently in a flush or flatorientation against a substrate using images from a macroscopic camera,and to identify and reorient any portion of the tissue of interest thatis not lying in such desired orientation against the substrate forproper imaging by the confocal microscope.

U.S. Pat. No. 7,864,996 describes a system for macroscopic and confocalimaging of in-vivo tissue having a macroscopic imager for capturing amacroscopic image of the tissue's surface for tissue, and a confocalimager for capturing one or more optically formed sectional microscopicimages on or within tissue. This system is for imaging in-vivo tissue ofa patient, such as skin, and uses a tissue attachment device onto apatient into which the macroscopic imager and confocal imager are eachseparately received. While useful, the tissue attachment is not designedfor use with a specimen of ex-vivo tissue which may be excised from apatient and placed on a substrate. U.S. Pat. Nos. 5,836,877 and6,684,092 describe a system having digital camera and a confocal imagerdirected to imaging tissue along the surface of the body of a patient.Like U.S. Pat. No. 7,864,996, the systems of U.S. Pat. Nos. 5,836,877and 6,684,092 are also designed for imaging in-vivo, rather than ex-vivotissue, which may be excised from a patient and placed on a substratefor optical sectional imaging.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide asystem and method for macroscopic and microscopic imaging of ex-vivotissue where images from a macroscopic imager can be used to providetissue orientation verification for imaging by the microscopic imager.

It is a further object of the present invention to provide a system andmethod for macroscopic and microscopic imaging of ex-vivo tissue whereprior to microscopic imaging, the tissue is verified as being in anorientation at least substantially flush against a surface of thesubstrate by being in focus in displayed macroscopic images prior toimaging by the microscopic imager, and if needed, any portion of thetissue unfocused is manually positioned until desired tissue sampleorientation is achieved.

It is still a further object of the present invention to provide asystem for macroscopic and microscopic imaging of ex-vivo tissue wheremacroscopic and microscopic imagers are each mounted in a common housingalong with a stage for moving the tissue with respect to the imagers.

Briefly described, the present invention embodies a system having amacroscopic imager, a microscopic imager, and a stage for moving asubstrate having optically transparent material supporting ex-vivotissue with respect to each of the macroscopic imager and themicroscopic imager to enable the macroscopic imager to capture one ormore macroscopic images, and the microscopic imager to capture one ormore optically formed sectional microscopic images on or within theex-vivo tissue, when presented to the ex-vivo tissue, via the opticallytransparent material of the substrate. A computer system controlsmovement of the stage with respect to the macroscopic imager and themicroscopic imager, and receives the one or more macroscopic images, andthe one or more microscopic images. A display is provided for displayingthe macroscopic images and microscopic images when received by thecomputer system.

Preferably, a housing contains at least the macroscopic imager, themicroscopic imager, and the stage. Such housing has a cover for blockingambient light when at least microscopic images are captured by themicroscopic imager.

The stage moves the ex-vivo tissue disposed upon the substrate along xand y orthogonal axes. Optics of the macroscopic imager for imaging theex-vivo tissue, and at least an objective lens of optics of themicroscopic imager for imaging the ex-vivo tissue, each have an opticalaxis oriented to extend at least approximately parallel with a z axisorthogonal to the x and y orthogonal axes. The imagers are each in adifferent assembly fixed in position in the housing with respect to thestage in accordance with the x, y, and z orthogonal axes prior to theex-vivo tissue being presented to the imagers.

The macroscopic images and microscopic images are each two-dimensionalimages spatially aligned with the x and y orthogonal axes of the stage.Thus, one of the macroscopic images of the ex-vivo tissue specimencaptured may be presented on the display and used to guide the selectionof one or more locations for capture of microscopic images by themicroscopic imager. This may be facilitated by overlaying one or moregraphical elements upon such macroscopic image indicating a location ofimaging by said microscopic imager with respect to said ex-vivo tissuedisplayed, and preferably microscopic imager's field of view relative tothe ex-vivo tissue.

The computer system captures macroscopic images from the macroscopicimager prior to the stage moving the ex-vivo tissue sample for imagingby the microscopic imager. During imaging by the macroscopic imager,macroscopic images of the ex-vivo tissue are displayed to verify thatthe tissue lies at least substantially flush or flat against the uppersurface of the substrate by being in focus in such images, and anyportion of the tissue specimen appearing unfocused in the microscopicimages on the display is manipulated upon the substrate until disposedagainst the substrate by being in focus in macroscopic images prior tothe stage moving the tissue sample for imaging by the microscopicimager. This avoids the problem of having tissue areas lying in anorientation that are not sufficiently planar and hence not available forproper imaging by the microscopic imager. The manipulation of the tissuespecimen may be carried out with the use of an external tool to manuallyposition unfocused parts of the tissue by an operator until such partsare displayed in focus when imaged by the macroscopic imager.

The microscopic imager is preferably a confocal microscope. Oneadvantage of the present invention over prior art for confocalmicroscopic imaging of ex-vivo tissue samples is that use of macroscopicimages for verification of the tissue sample orientation assures thatportions or areas of the ex-vivo tissue sample, particularly the outeredges which may be non-planar to a substrate when first present thereto,or portions of the tissue folded over each other or have air bubbles,are properly oriented for imaging, and if needed manipulated to a properorientation, against a substrate supporting the tissue specimen beforethe tissue sample is imaged by the confocal microscope via thesubstrate.

After verification (and manipulation if needed) of tissue sampleorientation, a cover substrate, device, or member may be applied andretained over the substrate providing a supporting base for the ex-vivotissue sample to retain such tissue sample orientation whenmicroscopically imaged. The cover substrate may optionally be so placedbefore the verification of tissue sample orientation using macroscopicimages. If the cover substrate is placed before such verification, thecover substrate may need to be temporarily removed to enable access tothe tissue sample for any needed manipulation as described above, andthen placed back thereupon the tissue specimen and its supportingsubstrate. Further, the cover substrate placement can serve as theexternal tool, or an additional tool, by providing a downward force tomanipulate the tissue specimen so that any non-planar portions moveagainst the tissue sample supporting substrate.

The present invention also embodies a method for macroscopic andmicroscopic imaging ex-vivo tissue comprising the steps of: mounting ina common housing at least a macroscopic imager, a microscopic imager,and a stage; placing ex-vivo tissue on a substrate having opticallytransparent material; mounting the substrate on the stage, which ismounted in the housing to move the substrate with respect to each of themacroscopic imager and the microscopic imager, wherein the substratemounting step is carried out before or after the placing step; movingthe stage to present the ex-vivo tissue on the substrate to themacroscopic imager; capturing, with the macroscopic imager, one or moremacroscopic images via the optically transparent material of thesubstrate; moving the stage to present the ex-vivo tissue to themicroscopic imager; capturing, with the microscopic imager, one or moreoptically formed sectional microscopic images on or within the ex-vivotissue; and displaying such one or more macroscopic images and one ormore microscopic images when captured with the aid of a computer systemreceiving such one or more macroscopic images and one or moremicroscopic images.

The capturing of one or more macroscopic images may further comprise thesteps of verifying the ex-vivo tissue lies at least substantially flushagainst a surface of the substrate by being in focus in the one or moremacroscopic images, and manually positioning any portion of the ex-vivotissue unfocused in the one or more macroscopic images on the displaysubstantially flush against the surface of the substrate until being infocus in the one or more macroscopic images. Positioning a cover may becarried out for blocking ambient light when at least one or moremicroscopic images are captured.

The capturing of macroscopic images may further comprise the step ofselecting one of the one or more microscopic images captured, and thedisplaying step displays the selected one of the microscopic images withdisplay of the one or more microscopic images when captured, andoverlays one or more graphical elements on the selected one of the oneor more macroscopic images indicating a location of imaging by themicroscopic imager with respect to the ex-vivo tissue displayed in theselected one of the macroscopic images to guide in selection of one ormore locations of the ex-vivo tissue for imaging by the microscopicimager.

While preferably the microscopic imager is operative by confocalmicroscopy, other optical sectioning microscopes may be used to providethe microscopic imager, such as those operative by two-photon microscopyor optical coherence tomography (OCT).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features, objects, and advantages of the invention willbecome more apparent from a reading of the following description inconnection with the accompanying drawings, in which:

FIG. 1 is a diagram of the system of the present invention with acomputer system for controlling and receiving images from macroscopicand microscopic imagers, which are mounted with a stage in a housinghaving a hinged cover;

FIG. 1A is a rear view of the housing of FIG. 1 having the macroscopicand microscopic imagers and stage showing connectors in which cables tothe computer system are removed;

FIG. 2A is a perspective view of the housing of FIG. 1 having themacroscopic and microscopic imagers and stage showing a specimenmounting platform for the stage movable with respect to optics of eachof the macroscopic and microscopic imagers in the housing, in which thecover of the housing is shown lifted to an up state;

FIGS. 2B and 2C are the same perspective view of the housing of FIG. 2Ashowing an example of an ex-vivo tissue sample when mounted upon asubstrate to the specimen mounting platform in which such stage is movedto present the tissue sample to the macroscopic imager in FIG. 2B andthen to the microscopic imager in FIG. 2C;

FIGS. 2D and 2E are broken perspective views of the specimen mountingplatform of FIGS. 2A and 2B with the upper wall of the housing removedshowing before and after, respectively, placement of a cover substrateupon a base substrate supporting a tissue sample to retain the tissuesample in its desired orientation after manipulation of one or moreportions to be substantially flush or flat against the base substrate,where FIG. 2D shows cover substrate prior to being manually positionedupon the base substrate;

FIGS. 3A and 3B are downwardly looking front and upwardly looking bottomexploded views, respectively, of the assembly of the macroscopic andmicroscopic imagers, stage, and specimen mounting platform in thehousing of FIG. 1, where completed assemblies of the macroscopic andmicroscopic imagers are shown and the cover of the housing is in alifted up state;

FIG. 4 is a cross-sectional view along line 4-4 in FIG. 1 of the housinghaving the macroscopic and microscopic imagers and stage in thedirection of arrows at the ends of such line;

FIG. 4A is a broken view of the cross-sectional view of FIG. 4 enlargedto show the optics of the macroscopic imager and part of the optics ofthe microscopic imager in more detail;

FIG. 5 is an exploded view of the assembly of the macroscopic imagerremoved from the housing of FIG. 1;

FIG. 6A is an exploded view of the microscopic imager removed from thehousing of FIG. 1;

FIG. 6B is an optical diagram of the optical system providing the opticsof the microscopic imager;

FIG. 7 is a perspective cross-sectional view of FIG. 1 along line 4-4 inFIG. 1 of the housing having the macroscopic and microscopic imagers andstage in the direction of arrows at the ends of such line showing thecover is in a lifted up state with an example of an ex-vivo tissuesample upon a substrate positioned by the stage for imaging by themacroscopic imager as shown in FIG. 2B, where the ex-vivo tissue samplecan be seen having at least one portion which is not lying flat againstthe substrate mounted to the stage;

FIG. 8 is an example of a screen of the display of the system of FIG. 1having a window for showing macroscopic images of the tissue sample ofFIG. 7 captured by the macroscopic imager, wherein portions of thetissue sample are unfocused and hence not positioned substantially flushor flat against the upper surface of the substrate;

FIG. 9 is the same perspective cross-sectional view of the housing ofFIG. 7 showing an example of an external tool for manipulating a portionof the ex-vivo tissue sample unfocused in the window of the displayshowing macroscopic images of the tissue sample, where such toolreorients such portion to lay substantially flush against the substratein a desired orientation for later microscopic imaging;

FIG. 10 is an example of the same screen of display of FIG. 8 having thewindow for showing macroscopic images of the tissue sample of FIG. 9captured by the macroscopic imager, verifying that the ex-vivo tissuesample is now in focus and hence lying substantially flush or flatagainst the upper surface upon the substrate in a sufficient properplanar orientation for microscopic imaging to be carried out;

FIG. 11 is the same perspective cross-sectional view of the housing ofFIGS. 7 and 9 in which the cover of the housing is placed in a downstate with the same example of an ex-vivo tissue sample of FIG. 2E,which is now retained between a cover substrate and the substrateproviding a base supporting the tissue sample, and positioned by thestage for imaging by the microscopic imager as shown in FIG. 2C; and

FIG. 12 is an example of the same screen of display of FIG. 10 havinganother window for showing microscopic images of the tissue sample ofFIG. 11 captured by the microscopic imager, a macroscopic still imagepreviously captured by the macroscopic imager of the same tissue sample,and graphical elements, such as a box and orthogonal cross-hair lines,overlaid upon such still macroscopic image showing the location andfield of view of the microscopic imager relative to the tissue sample.

DETAILED DESCRIPTION OF INVENTION

Referring to FIG. 1, a system 10 is shown having a housing 12 containinga macroscopic imager 14, a microscopic imager 16, and a stage 18, wherestage 18 moves a tissue specimen mounting platform 20 with respect toimagers 14 and 16, and in particular with respect to each of theiroptics 86 and 74, respectively, that enable imaging. As the cover 13 ofhousing 12 is shown closed, macroscopic imager 14, microscopic imager16, and stage 18 are blocked from view and shown in FIG. 1 by blocks indashed lines. Cover 13 is connected by a hinge 19 along housing 12 formoving the cover 13 between an up or open state and a down or closedstate, as denoted by arrows 21 in FIG. 2A. FIGS. 2A-C show cover 13lifted by a user to an up or open state, and FIGS. 1 and 1A show cover13 returned to a down or closed state.

A computer system 22, such as personal computer or workstation,communicates with each of imagers 14 and 16 via cables to ports 24 (FIG.1A) along an opening 26 in the back of housing 12 to control theoperation of imagers 14 and 16 and to receive signals representative ofimages from imagers 14 and 16. Images received from imagers 14 and 16can be stored in memory (such as RAM or hard drive) of computer system22, and outputted in either still or video formats to a display 23connected to computer system 22. A graphical user interface is providedon the screen 27 of display 23, as will be described later in connectionwith FIGS. 8, 10, and 12. Computer system 22 is also connected forcommunication with stage 18 via a cable to a port 25 in opening 26 ofhousing 12 to provide signals controlling movement of stage 18, wherebysuch signals can be sent to x and y motors of stage 18 to move the stagealong x and y orthogonal axes, respectively. User interface devices arealso connected to computer system 22, such as a keyboard 28 and mouse29, enabling a user to interact with the software or program operatingon computer system 22 to control operation of stage 18, macroscopicimager 14, and microscopic imager 16. Display 23 may be a touchscreendisplay which provides an additional user interface device to softwareoperating on computer system 22. An optional joystick 30 may beconnected by a cable to either a port on the computer system 22, orstage 18, to move stage 18 by providing signals to control x and/or ymotors of the stage 18 responsive to movement of the joystick by a user.

Referring to FIGS. 2A-E, and 3A-B, housing 12 is a generally rectangularcontainer having a top or upper wall 32 with a rectangular opening 33for accessing specimen mounting platform 20. Specimen mounting platform20 is insert-mounted in an opening 34 along the upper portion 18 a ofstage 18, as best shown in the exploded views of FIGS. 3A and 3B.Specimen mounting platform 20 has a rectangular aperture or opening 35having sides extending parallel to the x and y axes. Two arms 36 extendparallel to the y axis across aperture 35. Each of the arms 36 has twoopposing ends 37 with mounting pins 38, via holes in such ends, receivedin two slots 39 in the platform 20 that extend parallel to the x axis.Arms 36 each have inward facing recess or groove 40 shaped to receiveone of opposing ends 43 of a substrate 42 providing a base supportingtissue having a planar or flat upper surface 45 a. The example ofsubstrate 42 shown is of a typical plastic or glass slide as used withoptical microscopes. Recesses 40 may be shaped to receive a differentsubstrate of same or different size or thickness, or a specimen holderor cassette, such as described in U.S. Pat. No. 6,411,434, or otherdevice for holding a tissue sample. The specimen mounting platform 20without substrate 42 is shown in FIG. 2A, and with substrate 42 in FIGS.2B and 2C with an example of an ex-vivo tissue sample or specimen 44mounted thereupon the upper surface 45 a of substrate 42. Ex-vivo tissuesample 44 may be non-histologically prepared tissue removed from apatient/subject, such as from Mohs surgery, or other ex-vivo tissueexcised or otherwise acquired from a human or animal. Thus, the tissuespecimen shown in the figures is illustrative as the tissue specimen maybe of different shape, size, or thickness.

Preferably, as shown in FIGS. 2D and 2E, a cover substrate 46 is placedupon the substrate 42 providing a base supporting tissue sample 44 toapply a downward force upon the tissue sample against upper surface 45a. FIG. 2D shows cover substrate 46 prior to placement upon substrate42, and FIG. 2E after such placement. An adhesive 47 is present betweensubstrates 42 and 46 on opposite sides of tissue sample 44 near ends 43of substrate 42 to retain tissue sample 44 in a desired orientationagainst surface 45 a when imaged by microscopic imager 16. As will bedescribed later below, a tool 48 can be used to manipulate any portionof tissue sample 44 not sufficiently planar or flush against surface 45a of substrate 42 for proper imaging by microscopic imager 16 as part oftissue sample orientation verification. While preferably suchmanipulation is performed before placement of substrate 46 ontosubstrate 42, the adhesive 47 until set allows substrate 46 to be liftedaway from substrate 42 if needed to reorient any portions of the tissuesample 44, and then placed back upon substrate 42. For example, adhesive47 may be a liquid adhesive material applied just prior to placement ofsubstrate 46 upon substrate 42, or an optionally adhesive coatingprovided along one or both of upper surface 45 a of substrate 42 or thebottom surface of substrate 46, to join the substrates with tissuesample 44 there between. Substrate 46 may be identical to substrate 42,but a different device or member than substrate 46 may be used to applya downward force upon the tissue sample 44 against upper surface 45 a ofsubstrate 46. For purposes of illustration, substrate 46 is not shown inFIG. 2C.

One of arms 36 is fixed by its pins 38 in position along slots 39, whilethe other of arms 36 is movable along slots 39. The movable arm 36 ismoved by pushing pins 38 and sliding the arm along slots 39 to adjustthe distance with respect to the fixed arm 36 as needed so that the ends43 of substrate 42 are captured or retained in recesses 40 of arms 36.Releasing pins 38 of the movable arm 36 then retains its position alongaperture 35. Other mechanisms may be used along platform 20 for mountingsubstrate 42.

Once the substrate 42 is properly mounted to the stage 18, and tissuesample 44 is placed upon substrate 42, substrate 42 is moved by stage18, if not already in position, to first present the tissue sample 44 tooptics 86 of the macroscopic imager 14 as shown in FIG. 2B for capturingone or more macroscopic images, and then substrate 42 is moved by stage18 to present the tissue sample 44 to an objective lens 72 of optics 74of the microscopic imager 16 as shown in FIG. 2B for capturing one ormore microscopic images. When stage 18 presents tissue sample 44 uponsubstrate 42 to each imager 14 and 16, images are captured by the imagerthrough the optical transparent material of the substrate 42 via boththe lower bottom surface 45 b and upper surface 45 a of substrate 42, asshown in FIGS. 7 and 11, respectively. While preferably substrate 42 isentirely of transparent material, such as glass or plastic, only suchportion upon which tissue sample 44 is disposed need be of transparentmaterial to the illumination to imagers 14 and 16 to enable imagingthrough substrate 42.

Cover 13 in its down state over upper wall 32 blocks (or at leastminimizes) ambient light from being received by imagers 14 and 16,respectively, when each are operated to capture images. Cover 13 isoptional where system 10 is utilized in environments where the level ofartificial/natural lighting can be controlled so that ambient light isminimized during operation of imagers 14 and 16.

Referring to the exploded views of FIGS. 3A and 3B, and cross-sectionalview of FIGS. 4A and 4B, the assembly of stage 18, and imagers 14 and16, in housing 12 is shown. Housing 12 has a generally rectangular caseor casing 50 having fours sides 52 a and an attached bottom 52 b. Sides52 a attach to bottom 52 b by screws 53 which extend through holes infour flanges 54 a into threaded holes 55 along corner posts 54 b inbottom 52 b that extend upward to meet flanges 54 a. Case 50 has anupper opening 56 having a recessed ledge 57 upon which upper wall 32 isattached by screws 58 (FIGS. 2A-C), via holes 59 in upper wall 32, intothreaded holes 60 along the ledge 57. Sides 52 a and bottom 52 b of case50 may be contoured as shown in the figures, but another case may beused to provide housing 12. Along bottom 52 b of case 50 are feet 61 forsupporting housing 12 upon a surface, such as a table.

Prior to assembly of case 50, a base plate 62 is attached to bottom 52 bby screws 63 via holes in base plate 62 into threaded holes along bottom52 b. Two parallel vertical walls 64 attach by screws 65 a to threadedholes 65 b along base plate 62 for supporting stage 18. Stage 18 may bea typical two-dimensional translation stage having an upper portion 18 athat moves along the x axis, and a lower portion 18 b that moves alongthe y axis. For example, stage 18 may be a Marzhauser X-Y StageScan^(Plus) Model No. 00-24-579-0000 (Manufacturer: Märzhäuser WetzlarGmbH & Co. KG, Germany). A stage controller card may be provided in thecase for computer system 22 to enable such interface with stage 18. Inthe case of Marzhauser X-Y Stage, such stage control card may be aScan^(Plus) Marzhauser X&Y Stage Controller Card, Part Number00-76-150-0813. Stage 18 is mounted along the top of vertical walls 64as typically in mounting stages to a base, such as by screws 66 viaholes in two stage mounts 67 into threaded holes 68 along top ofvertical walls 64.

Microscopic imager 16 is mounted to an upper plate 71 a of a stage 70.The base 71 b of stage 70 is attached to base plate 62 so that theobjective lens 72 of microscopic imager 16 is disposed to extend upwardsinto an opening 17 of stage 18, and may extend at least partially intoaperture 35 of platform 20 if needed to present objective lens adjacentbottom surface 45 b of substrate 42. Base 71 b is attached to base plate62 by screws 75 into threaded holes 76 along base plate 62. In stage 70,upper plate 71 a is movable with respect to base 71 b in tip, tilt androtation using micrometers 71 c attached to internal gearing thatcouples upper plate 71 a to base 71 b in order to align the optical axis73 of objective lens 72 to extend along a z axis orthogonal to the x andy axes along which stage 18 moves, and to rotate plate 71 a so that theraster scan line of images captured by imager 16 is finely adjusted withmovement of stage 18 along its y axis, as will be described later. Forexample stage 70 may be a Model No. TTR001-Tip, Tilt, & Rotation Stagemanufactured by Thorlabs, Inc., but other stages may be used. Aprotective cover 78 is disposed over imager 16 and stage 70 so thatobjective lens 72 extends through an opening 79 in cover 78. Cover 78may be attached by screws in the base of cover 78 to base plate 62. Acable 80 from electronics of imager 16 extends via another opening incover 78 to a connector providing one of ports 24 mounted to opening 26in case 50 providing the back of housing 12.

Microscopic imager 16 is preferably a confocal microscope for capturingoptically sectioned images, such as used in the VivaScope® sold byCaliber Imaging and Diagnostics, Inc. of Rochester N.Y., USA. Suchconfocal microscope can capture microscopic sectional images of thetissue specimen at different depths within the tissue sample 44 and atthe surface of such tissue sample 44 that lies against upper surface 45a of the substrate 42, from scanned illumination of one or more lasersfocused and collected via objective lens 72 of optics 74. Movement ofstage 18 enables automatic or manual selection of a particular locationalong the ex-vivo tissue sample 44 in terms of x and y coordinates, inwhich depth of the section on or in the tissue specimen is selected bymoving an objective lens 72 along optical axis 73 aligned along the zaxis of system 10 orthogonal to the x and y axes of stage 18 using stage70.

The optical system providing optics 74, electronics of microscopicimager 16, and operation of computer system 22 with display 23 may bethe same as described in U.S. Pat. No. 9,055,867, and preferably is thesame as the imaging head of the confocal microscope of pending U.S.patent application Ser. No. 15/810,093, filed Nov. 12, 2017, which areboth incorporated herein by reference. U.S. patent application Ser. No.15/810,093 describes in detail the optical components and electronics ofthe imaging head of imager 16 being mounted along a chassis 176 andsupport plate 177, as shown and described in connection with FIGS. 9,10, and 11 of the incorporated U.S. Patent Application.

The particular application of the confocal microscope of U.S. patentapplication Ser. No. 15/810,093 as incorporated in system 10 isdescribed below in reference to FIGS. 4, 4A, 6A, and 6B. Two printedcircuit boards 190 with electronics for controlling the confocal imaginghead of microscopic imager 16, responsive to computer system 22, areattached to chassis 176, where circuit boards 190 are connected to othercircuit boards described herein. While FIG. 6A shows an exploded view ofthe microscopic imager 16 assembly shown in FIG. 4, the optical diagramof FIG. 6B best illustrates the operation of the optical systemproviding optics 74 of microscopic imager 16 for enabling imaging. Afirst laser illumination source 220 provides a linearly polarized beam221 at a single wavelength (e.g., 488 nm), and a second laserillumination source 223 provides a linearly polarized beam 224 at adifferent single wavelength (e.g., 785 nm). Beam 221 is combined withbeam 224 into a beam 226 by a dichroic filter 225. If desired, anoptional third laser illumination source 227 may be provided producing alinearly polarized beam 228 at a different discrete wavelength than thatof beams 221 and 224 (such as at 405 nm, 561 nm, or 640 nm). If lasersource 227 is present, beam 226 combines with beam 228 by a dichroicfilter 229 to provide beam 230. Laser sources 220, 223 and 227preferably are laser diodes received and retained in cylindrical tubes220 a, 223 a, and 227 a, respectively, each with an opto-detector formonitoring their laser power, and then mounted with filters 225 and 229in a structure or block 178, such as of aluminum, which is mounted tochassis 176.

Beam 226 (or beam 230 if source 227 is present) passes through apolarizing beamsplitter 231. A resonant scanner 232 presents itsscanning mirror 232 a to beam 226 (or beam 230 if source 227 ispresent), and the beam from the resonant scanner mirror 232 a is thenincident scanning mirror 234 a of a galvanometer 234 to provide a scanbeam 236. Mirrors 232 a and 234 a oscillate so that mirror 232 a fast orhorizontal line scans in a raster being scanned, and slow or verticalscan and retrace are provided by mirror 234 a, as described in theincorporated U.S. Pat. No. 9,055,867. The axes of oscillation of thesemirrors 232 a and 234 a are orthogonal (perpendicular) to each other.The separation distances may be approximately a minimum separationdistances to provide clearance between the mirrors as they scan. Atelescope 238 magnifies (e.g., 2.3 x) and relays scanning beam 236 toobjective lens 72 via a quarter wave plate shifter 239, and theobjective lens 72 focuses the scanning beam 236 to the tissue sample 44being imaged on substrate 42 when stage 18 presents the scanning beam236 to a location along such tissue sample 44.

The return light 240 from the tissue sample 44 passes through objectivelens 72, quarter wave plate 239, telescope 238, and scanning mirrors 232a and 234 a. The return light thus is descanned by mirrors 232 a and 234a into a stationary beam 241 and enters the polarizing beamsplitter 231which reflects beam 241 via a focusing lens 242, a reflecting mirror243, and a notch filter 244, to a dichroic beamsplitter 245 which splitsthe returned light into a first beam 246 and a second beam 250. Beam 246is incident a small aperture provided by pinhole 247 onto a detector 249via one of selectable open or filter positions along a filter wheel 248.Beam 250 is incident a small aperture provided by a pinhole 251 onto adetector 253 via one of selectable open or filter positions along afilter wheel 252. Each filter wheels 248 and 252 has a motor 174 and 175which can move the wheel to select a position of a desired opticalfilter or opening (if present) along the wheel. Detectors 249 and 253may each be provided by a photomultiplier tube on circuit boards 182 and183, respectively, which are then mounted to support plate 177. Lens 242focuses returned light beam 241 split into beams 246 and 250 ontorespective pinholes 247 and 251. Although not shown in FIG. 6B, aturning mirror 173 may be provided between beamsplitter 231 and mirror232 a to reflect beam 226 (or beam 230 if source 227 is present) ontomirror 232 a, and reflects beam 241 from mirror 232 a to beamsplitter231. Electrical signals outputted from each of detectors 249 and 253 arereceived by electronics of the imager 16 to provide video signals, timedin accordance with fast and slow scan mirrors 232 a and 234 a,representative of sectional microscopic image on or in tissue sample 44for communication to computer system 22 as described in incorporatedU.S. Pat. No. 9,055,867.

As the illumination of the tissue sample 44 is of multiple discretewavelengths in accordance with laser sources 220, 223, or 227, detectors249 and 253 receive different wavelengths of the collected illuminationbeam 241 from objective lens 72 to enable simultaneous capture of a sameone of the microscopic images at the different wavelengths or wavelengthrange on detectors 249 and 253 in accordance with selected position ofone of optical filter or an opening on filter wheels 248 and 252,respectively. Where one or more of the discrete wavelengths ofillumination can activate fluorescent dye(s) that may be applied totissue sample 44, a selected optical filter along wheels 248 and 252 ispositioned in the path of the beam to detectors 249 and 253 to enabledetection of fluorescent wavelength(s) associated with the dye(s) ontheir associated detector. Where non-fluorescent imaging is desired, aselected open position along wheels 248 and 252 is positioned in thepath of the beam to detectors 249 and 253 to detect a discretewavelength of illumination of the scanned illumination 236 to the tissuesample. Notch filter 244 allows selectable discrete wavelengths orranges of wavelengths to assist in detecting wavelengths with filtersalong the filter wheels. Preferably, notch filter 244 allows light ofwavelength of laser 223 (e.g. 785 nm), and blocks light of wavelengthswhich may interfere with imaging at fluorescent wavelengths associatedwith the filters disposed along filter wheels 248 and 252 in the path oflight for detection by their respective detectors 249 and 253. Due tothe perspective of the exploded angular view of FIG. 6A, not allcomponents of optics 74 shown in FIG. 6B can be seen in FIG. 6A. Also,while multiple laser sources and two detectors are preferred, a singlelaser source and single detector for detecting reflectance microscopicimages, such as at 785 nm wavelength, may be provided in optics 74.

The microscopic imager 16 differs from that shown in incorporated U.S.patent application Ser. No. 15/810,093 in that the support plate 177 isattached by screws into threaded holes along upper plate 71 a of stage70, and the path of scanned illumination 236 focused by, and collectedreturned light 240 from, objective lens 72 is reflected by a fixedmirror 237 in a fixture attached by a tube or barrel 255 by screws 256into threaded holes along the front of chassis 176 so such path can thenextend along optical axis 73 of objective lens 72 when aligned along thez axis of system 10, as described earlier using stage 70. Objective lens72 is disposed at the upper end of a barrel 257 providing a tube orsleeve moving axially (along optical axis 73) by a linear actuator, asdescribed in the incorporated U.S. Pat. No. 9,055,867, over a fixed tube258 attached to a plate 168. Objective lens 72 may be moved along theoptical axis 73 (extending parallel to the z axis of system 10) so as tofocus the beam at selected locations at the surface or the internalsections of the specimen to be imaged. The quarter wave plate 239 isretained in a holder 260 mounted in end of barrel 257 receivingobjective lens 72. Lenses in barrel 255 and fixed tube 258, along thescanning illumination 236 and collected returned light 240 paths bent 90degrees by angled mirror 237, provide telescope 238. A magnetic strip isprovided on the side of barrel 257 which is read by a sensor 170 thatlinearly encodes position of the barrel 257 to the electronics of imager16, thereby enabling computer system 22 to actuate a linear motor 161 toadjust the position of objective lens 72 with respect to telescope 238and hence the focus of such lens 72 with respect to the tissue sample44.

While components providing multiple wavelengths of laser illumination asdescribed in the incorporated U.S. Patent Application may be used,preferably two laser sources 220 and 223 of wavelength 485 nm and 785nm, respectively, are provided in microscopic imager 16, however,different wavelength lasers, or additional laser(s) may be used, such asby providing laser source 227. Power and ground to electronics and othercomponents in imager 14 and 16 and stage 18 are provided by wires withincables to ports 24 and 25 along housing 12. Other optical sectioningmicroscopes may provide microscopic imager 16, such as those operativeby optical coherence tomography (OCT), or two-photon laser microscopy.

To enable faster image acquisition by imager 16, computer system 22 canfix the position of galvanometer mirror 232 a, and instead move stage 18in a stepwise fashion along the y axis to provide comparable raster scanimaging. It has been found however unless the raster scan lines followsprecisely travel of the stage 18 moving along the y axis, successiveraster image lines will not align in each frame of the microscopicimages. To solve this problem, a calibration target with crosshair orother reticle lines representing a full frame is located in tissuespecimen mounting platform 20 in a same position as substrate 42 (thussuch crosshairs align parallel with x and y axes of stage 18 motion),and stage 18 moved to present such target to objective lens 72 so thatmicroscopic images of the target are shown on display 23. Stage 70 isrotated by one of micrometers 71 c (or other ones of micrometers 71 c ifneeded) until a full frame is captured without any breaks in thecaptured image displayed, thus aligning stage 18 movement along the yaxis with the raster scan line. Stage 18 repeats movement in a firstdirection than in the reverse direction along the y axis as a full frameis captured, and in each pass along the first direction follows the samedirection of raster scan line of mirror 232 a albeit faster in speedthan mirror 234 a. Optionally, the raster scan lines can be captured inboth first direction and in the reverse direction to further increasethe acquisition time of each frame.

Each filter wheels 248 and 252 has a motor 174 and 175 driven byelectronics on a printed circuit board 185 to select a position of oneof multiple optical filters or an opening desired along each filterwheel for light returned on their respective detectors 249, and 253, viatheir respective pin holes 247 and 252. For each wheel, the printedcircuit board 185 has a Hall effect sensor which reads a magnet alongthe wheel to sense the home position of the wheel and rotate the wheelto the desired filter or open location along the wheel by actuationsignals received from computer system 22 as stored in its memory.Circuit board 185 for driving and controlling motors 174 and 175 may besupported on circuit board 182. A structure or block 180, which may beof aluminum, is mounted to chassis 176 to support filter wheels 248 and252, pinholes 247 and 252, beamsplitter 245, and notch filter 244, forimaging onto such detectors 249 and 253 as described earlier. Pinholes247 and 251 (i.e., each provided by a thin substrate with light blockingmaterial having a small aperture) may have components shown in FIG. 6Afor mounting in structure 180. In order to properly align beams 246 and250 for detection on respective detectors 249 and 253, mirror 243 andone or more of pinholes 247 and 251 may each be adjustable in position.For example, pinhole 247 may be mounted in a cylinder, prior to beingmounted in structure 180, and positionally adjustable in such cylinderusing set screws with respect to beam 246, and mirror 243 mounted uponan adjustable flexure attached to a bracket or flange 186 of chassis 176for steering beam 250 via beamsplitter 245, so that both beams 246 and250 are aligned for detection. Pinhole 251 may optionally be similarlyadjustable in position.

Referring to FIGS. 3A, 3B, 4, and 4A, macroscopic imager 14 is shownhaving a digital camera 82 with two circuit boards 84 for presenting atwo-dimensional detector array 85 of pixel sensing elements, such as aCCD, for capturing two-dimensional color images aligned with the x and yaxes of stage 18, via optics 86 that focus along an optical axis 83 ator near the plane of the upper surface 45 a of substrate 42 when stage18 is positioned to present substrate 42 to imager 14. The circuitboards 84 of the digital camera 82 are attached to a mounting plate 87that attaches, via a cylindrical coupling member 97, to a base 88, whichis mounted by screw 89 into threaded holes 90 (FIGS. 3A and 3B) alongbase plate 62. Macroscopic imager 14 has an illumination system 96 whichilluminates aperture 35 of platform 20 to provide light to enable propercapture of macroscopic images by imager 14 of tissue sample 44.Macroscopic imager 14 and its illumination system 96 are described inmore detail below in connection with FIG. 5. A protective cover 93 isdisposed over circuit boards 84 with an opening 92 through which isextended a barrel 94 supporting optics 86 disposed over the array 85 ofsensing elements upon the uppermost one of circuit board 84. An “L”shaped plate 98 is attached between vertical walls 64 by screws receivedvia holes 99 in walls 64 into threaded holes 100 along opposite ends ofplate 98. Plate 98 has a vertical portion 102 a and a horizontal portion102 b with an opening 101 through which extends barrel 94 of macroscopicimager 14 after passing through opening 92 of cover 93 (FIGS. 4A and4B).

Referring to FIG. 5, an exploded view of the assembly of the macroscopicimager 14 is shown. Macroscopic imager 14 has a digital camera 82, suchas Model No. LW570, sold by Lumenera Corporation of Ottawa, Ontario,Canada. Digital camera 82 is an assembly of two circuit boards 84 witharray 85 of sensing elements and electronics enabling high resolution,such as 5 Megapixel, color images. Unlike microscopic imager 16, digitalcamera 82 provides a larger, macro view of the ex-vivo tissue sample 44without microscopic resolution. A cable 104 from electronics of imager14 provided by digital camera 82 extends via an opening 92 in cover 93to a connector providing another one of ports 24 mounted in opening 26of case 50.

The optics 86 of macroscopic imager 14 comprise a relay lens 106 and aplano-concave lens 107 which are contained in barrel 94. Barrel 94 hasan opening 108 with an upper end 110 a of a diameter for receiving theplano-concave lens 107, and a lower end 110 b of a diameter and lengthfor receiving relay lens 106. To retain each of the lens 106 and 107 inbarrel 94 are two externally threaded retainer rings 111 a and 111 b,which after placement of O-rings 112 a and 112 b to upper and lower end110 a and 110 b, respectively, of opening 108 engage threads along theentrance into such upper and lower end 110 a and 110 b, respectively, ofopening 108. Preferably, a liquid adhesive is applied to fix rings 111 aand 111 b in position after engaging barrel 94. Barrel 94 may be mountedto upper one of circuit board 84 over array 85 by two screws into holeson either side of array 85. Mounting plate 87 is attached to circuitboards 84 by screws 114 extending, via holes 115, in mounting plate 87into threaded holes of four standoffs 116 having threaded ends thatscrew into threaded holes into four standoffs 117 that couple thecircuit boards 84 together. Plate 87 extends to a lower cylindrical end87 a with external threads which engage upper interior threads ofcylindrical coupling member 97. Coupling member 97 has lower interiorthreads which engage threads along the upper cylindrical end 88 a ofbase 88. To adjust the focus of macroscopic imager 14, a target withcrosshair or other reticle line(s) is located on tissue specimenplatform 20 in the same position as substrate 42 (thus such crosshairsor reticle lines align parallel with x and y axes of stage 18 motion) sothat a macroscopic image (or images, such as video) thereof is shown ondisplay 23, and coupling member 97 is rotated clockwise orcounterclockwise to raise or lower plate 87 until the target is in focuson display 23. When focus is achieved, set screws 118 which extendthrough threaded holes 119 along coupling member 97 and/or base 88 tofix the coupling member 97 in position. The target may be used furtherto assure that the two-dimensional images captured by array 85 at leastapproximately align with x and y axes of stage 18. With plate 87 socoupled to housing 12, optics 86 and array 85 of macroscopic imager 14are so fixed in position in housing 12 so that two-dimensionalmacroscopic images captured by array 85 extend parallel with x and yaxes of stage 18, and optical axis 83 extends at least approximatelyparallel with the z axis orthogonal to the x and y axes of system 10.Thus, as optical axes 73 and 83 are oriented parallel to the z axis ofsystem 10, they are at (or at least approximately) co-axial with eachother.

Illumination system 96 is provided by LEDs 120 mounted in a ring alongan annular circuit board 122 having a central opening 124, and usesreflective surfaces 127 and 133 to reflect light from the LEDs 120upwards without directly being incident tissue sample 44 disposed onsubstrate 42. This is achieved by a cylindrical member 126 havingcircular symmetric parabolic reflective surface (or reflector) 127 whichincreases in diameter as it extends upwards from a central aperture 128into which extends the upper end of barrel 94, thereby aligningparabolic reflective surface 127 along optical axis 83. Received incylindrical member 126 is a truncated conical member 130 having a lowerend 132 and a central opening 131 extending through member 130, vialower end 132, along optical axis 83. The lower end 132 is mounted intoupper end of barrel 94 after extending through central opening 124 ofcircuit board 122 in order to mount the circuit board 122 at the base ofparabolic reflective surface 127. The truncated conical member 130 hasan outer conical reflective surface 133 which increases in diameter fromlower end 132 until it nears the height of cylindrical member 126.Illumination produced by the light sources provided by LEDs 120 passesinto a gap 134 between conical reflective surface 133 and parabolicreflective surface 127 and exits upwards along a path 136 as indicatedby arrows in FIGS. 4 and 4A. While reflective surface 133 may be smooth,reflective surface 127 is preferably unfinished and less smooth thansurface 133 and at least partially diffuses light before exiting upwardsin gap 134 along path 136 about the circumference of truncated conicalmember 130.

A ring of polarizer film 138 is disposed about the top of truncatedconical member 130 to extend over gap 134 so that light from LEDs 120(and/or reflected by surfaces 127 and 133) exiting gap 134 passesthrough polarizer film 138 to provide polarized light for use inilluminating tissue sample 44 when present over macroscopic imager 14.The polarizer film 138 is retained in position over gap 134 by acircular plate 140 and screws 141 that extend via holes 142 in plate 140into threaded holes 143 along the top of truncated conical member 130 sothat plate 140 clamps polarizer film 138 over gap 134. Plate 140 has acentral aperture or opening 144 with a downwardly extending cylinder 146mounted to plate 140 or part of such plate 140. Cylinder 146 extendsdownwardly from plate 140 into central aperture 131 of truncated conicalmember 130. A polarizing lens 148 is mounted in aperture 131 oftruncated conical member 130 prior to cylinder 146 being disposeddownward in central aperture 131. As the illumining light is polarized90 degrees by polarizer film 138 along the illumination path 136 totissue sample 44, returning light from tissue sample 44 is depolarizedby polarizing lens 148 before reaching array 85. The returning lightrepresentative of a macroscopic image of tissue sample 44 travels alongan imaging path from tissue sample 44 through aperture 144, cylinder146, polarizing lens 148, aperture 131 of truncated conical member 130,and central opening 124 of annular circuit board 122, and is imaged byoptics 86 in barrel 94 onto array 85 of sensing elements. Plate 140 andcylinder 146 are of black anodized plastic material, which improvesimaging by array 85 by blocking light along the imaging path which isnot associated with light representative of tissue sample 44.

Disposed over plate 140 is shown an optional tray 150. Tray 150 has anopening 151 aligned with aperture 144 of plate 140 and cylinder 146.Screws 141 extend into holes 153 along tray 150 to retain the tray inposition with objective lens 72 of imager 16 extended through an opening152 of tray 150. A cylindrical wall may extend upwards along opening 152to form a generally conical structure with an open top of a diameter forreceiving there through objective lens 72. The tray 150 is of opticallytransparent material, such as clear polycarbonate, to pass light fromillumination system 96 of macroscopic imager 14. While tray 150 isoptional, it extends under stage 18 to provide a mechanism for catchingany debris which may inadvertently fall via aperture 35 of platform 20,and further protects imagers 14 and 16.

The LEDs 120 of illumination system 96 are activated responsive to powerbeing supplied by a cable 145 (FIG. 5) from circuit board 122 to theelectronics of the digital camera 82 on circuit boards 84. LEDs 120 areenabled by such electronics responsive to signals received from computersystem 22 to activate camera 82, and power is disable to LEDs 120 whencamera 82 is not activated. Optionally, the user can toggle the LEDs 120of macroscopic imager 14 on and off as desired, and in response computersystem 22 communicates with such electronics to enable or disable theLEDs accordingly. For example, sixteen LEDs 120 may be used spaced in aring along circuit board 122. Such LEDs 120 may be white light sources,but other wavelengths of emitted light may be used depending on thesensitivity of array 85 sensing elements to returned light from theilluminated tissue sample 44 with light from LEDs 120.

Base plate 62, vertical walls 64, and plate 98 are of rigid material,such as stainless steel or aluminum. Case 50, upper wall 32, cover 13,protective covers 78 and 93, and tray 150 may be made of molded plasticmaterial. Also, a fan 154 is mounted onto base plate 62 by screws 155 inholes 156 along base plate 62, and powered by electronics on microscopicimager 16. While holes in base plate 62 are described above as beingthreaded for receiving their respective screws, such holes in base plate62 need not be threaded, and nuts may be then used to secure the ends ofsuch screws to base plate 62. Cylindrical member 126 and truncatedconical member 130 may also be formed from stainless steel or aluminum,or other rigid material, and if needed coated with a layer of reflectivematerial, such as aluminum.

As shown in FIGS. 4 and 4A, the optical axis 73 of objective lens 72 ofmicroscopic imager 16 are aligned along the vertical z-axis orthogonalto x and y axes of system 10 along which stage 18 is movable. Duringmanufacture, or for maintenance with removal of casing 50 and the plateproviding upper wall 32 from housing 12, a substrate having downwardfacing reflective surface along the xy plane of x and y axes of thestage 18, such as a mirror, is placed upon platform 20 in the samemanner as substrate 42 described earlier, and the stage 18 is positionedso that an image from microscopic imager 16 is shown on display 23.Micrometers 71 c of stage 70 are adjusted as need in the tip, tilt, androtation of upper plate 71 a of stage 70 until a maximum bright spotappears on display 23 to a technician indicating that the optical axis73 of imaging is perpendicular to the reflective surface. The substratehaving a reflective surface is then removed, and tilt adjustment iscomplete. As macroscopic imager 14 by virtue of its mounting in housing12 has been found not to require tilt or tip adjustment to align alongoptical axis 83 of optics 86 with z axis of system 10. Rather,adjustment of focus as described earlier provides sufficient alignmentfor imaging of tissue sample 44 by macroscopic imager 14. However, ifdesired, the same substrate used for alignment for microscopic imager 16may be used to verify that optical axis 83 is aligned parallel to z axisof system 10 by similarly viewing one or more macroscopic images of suchsubstrate on display 23.

The start x and y positions of stage 18 for each of macroscopic imager14 and microscopic imager 16 are determined using a target withcrosshairs or other reticle lines in tissue specimen mounting platform20 in place of substrate 42 which enable such crosshairs or reticlelines to align parallel with x and y axes of stage 18 motion. Stage 18first is positioned by a technician to provide one or more macroscopicimages to align the target with macroscopic imager 14 on display 23, inthe same manner as in focus adjustment described earlier. The computerstores in memory the x and y start position of the macroscopic imager14. Stage 18 is next positioned by the technician to provide one or moremicroscopic images to align the target with microscopic imager 16 ondisplay 23 both in x and y of stage, and along the z axis by movingobjective lens 72 along its optical axis 73 to determine zero depth. Thecomputer system 22 stores in memory the x, y, and z start position ofthe microscopic imager 16. The above calibration of microscopic imager16, and if desired of macroscopic imager 14, and setting of startpositions for use with such imagers, may be carried out by a technicianin system 10 using the same graphical user interface on display 23 asdescribed below in connection with FIGS. 8, 10, and 12.

Referring to FIGS. 2A-2E and 7-12, the operation of system 10 startswith tissue sample 44 being placed upon upper surface 45 a of substrate42 which is retained between arms 36 of tissue specimen mountingplatform 20 of stage 18, as described earlier and shown in FIGS. 2B and7. The tissue sample 44 may be placed on substrate 42 before or afterthe substrate in placed in a retained position on the tissue specimenmounting platform 20. If desired, the user has placed cover substrate 46over substrate 42, but typically such is not preferred until aftersample orientation verification, and any manual tissue repositioning ifneeded, is completed. Computer system 22 sends signals to the x and ymotors of stage 18 to move platform 20 to the stored start x and yposition in memory of computer system 22 centered with optical axis 83of macroscopic imager 14 onto tissue sample 44, and signals to theelectronics of macroscopic imager 14 to capture macroscopic images withLEDs 120 actuated. The cover 13 at this time may be in up or down state,as desired by the user.

Next, tissue sample orientation verification is performed as often outeredge(s) of tissue sample 44 and the tissue sample's surface facing uppersurface 45 a of substrate 42 are non-planar with respect to surface 45a. Such can be due to the tissue sample 44 being folded over each other,or air bubble(s) being present between tissue sample 44 and surface 45a, keeping the tissue from lying flat or planar upon surface 45 a. Inthe example of FIG. 7, a fold 44 a of the tissue sample 44 is present.

In FIG. 8, an example graphical user interface on screen 27 of display23 is shown having macroscopic images 199 captured by array 85 ofmacroscopic imager 14 displayed in a window 200 as video. Fold 44 a inthe image in window 200 appears out of focus (fuzzy) with the rest ofthe image, further while not readily apparent to the user, additionalportions or areas 44 b along tissue 44 are now seen that are also not infocus and hence not planar with upper surface 45 a of substrate 42.Areas 44 b may be due to non-planar side edges of the tissue sample orexcessive air bubbles. As shown in FIG. 9 with cover 13 in an up or openstate, a user manually locates an end of tool 48 to gently unfold thefold 44 a, and manipulate areas 44 b until the images 199 in window 200are in focus as shown in FIG. 10. Optionally, computer system 22 mayhave software which automatically processes the macroscopic images 199to determine presence of any unfocused areas of the images, andgraphically indicates any unfocused portions on the display of saidmacroscopic images, such as using arrows as depicted in FIG. 8, toassist the user.

Another view of tool 48 operating upon fold 44 a is shown in FIG. 2D. Asstated earlier, cover substrate 46 is applied upon substrate 42 toretain the desired orientation of tissue sample 44 against upper surface45 a of substrate 42. The downward pressure of cover substrate 46applied along the top of the tissue sample 44 can further keep anymanipulated non-planar side edges planar against surface 45 a. Theresulting substrate-tissue sample-substrate sandwich or carrier 49 isshown in FIGS. 2E, 4, and 11. Optionally, an index matching fluid isapplied the tissue sample 44 prior to placement of cover substrate 46 tominimize reflection and spherical aberration when later imaged bymicroscopic imager 16. Typically, the tissue sample 44 is stained andwashed with saline prior to being placed on substrate 42 without anindex matching fluid. If desired, such stain may be a fluorescent dyeapplied to the tissue sample 44 that can be activated by one of thelaser illumination sources of microscopic imager 16 in order to enhancetissue structures of interest. Less preferably, imaging by themicroscopic imager 14 is carried out without cover substrate 46.

If the macroscopic images of tissue sample 44 in window 200 of screen 27is not centered, the user via keyboard 28, joystick 30, or mouse 29clicking on graphical elements associate with stepwise motion of x and ymotors as desired. The images from imager 14 may be captured havingapproximately a 25 mm square field of view from array 85 of digitalcamera 82. By changing optics 86 and/or size of array 85, differentfields of view can be captured, as may be useful to capture largertissue samples than presented in the example of tissue sample 44.Optionally, the field of view may be electronically zoomed in or out byeither image processing of computer system using buttons 201 (or bysignals from the computer system 22 to the electronics of camera 82 ofmacroscopic imager 14 to provide a different resolution image ifavailable), as needed to select a different field of view which capturesthe entire tissue sample 44, or such part of tissue sample 44 ofinterest.

With the tissue sample 44 verified in this manner as being properlyoriented against upper surface 45 a of substrate 42, i.e., all (or areasof interest) are sufficiently planar or flush against surface 45 a, andany centering and/or zooming is completed, a picture of the tissuesample is captured by the user selecting with the mouse 29 a “TakePicture” button 201 on screen 27. A still macroscopic image 206, ratherthan a live video images from imager 14, is now displayed in window 200and stored in memory of computer system 22 for later use as a guide ormap when capturing microscopic images. Still macroscopic image 206represents one of such macroscopic images 199 captured by array 85. Ifthe still macroscopic image 206 is not acceptable to the user, the userselecting with the mouse 29 a “New Picture” button 203 on screen 27returns to the video of macroscopic images 199 from macroscopic imager14 in window 200, and then a new still macroscopic image 206 of tissuesample 44 can be captured using button 201 and stored in memory ofcomputer system 22. Also, a “Lights” button 204 can be selected by themouse 29 to toggle the LEDs 120 of macroscopic imager 14 on and off asdesired, since sufficient ambient light may be present when cover is upor open without the need for illumination of the tissue sample 44 usinglight from LEDs 120. The cover 13 may be in an open and up state whenimages are captured by macroscopic imager 14. However, the cover 13 maybe down state if desired when capturing still macroscopic image 206.

Next, a drop of index matching fluid, such as ultrasonic gel, is placedupon the upper tip of objective lens 72 by the user prior to movingstage 18 to the start position for microscopic imager 16. Such upper tipof objective lens 72 being accessible via aperture 35 (FIGS. 2B, 7 and9). Objective lens 72 is optically coupled by such fluid to bottomsurface 45 b of substrate 42 to minimize reflection and sphericalaberration as objective lens 72 is located at the start z position andmoves along bottom surface 45 b to one or more different locations uponor within tissue sample 44.

With cover substrate 46 placed upon the substrate 42, the tissue sample44 now verified as being substantially flush or planar upon surface 45a, and index matching fluid in placed on the upper tip of objective lens72 which will face bottom surface 45 b of substrate 42, computer system22, responsive to the user clicking on button 213 using mouse 29, sendssignals to x and y motors of stage 18 to move tissue specimen mountingplatform 20 to the stored start x and y position (as shown in FIGS. 2Cand 11) stored in memory of computer system 22 for the microscopicimager 16 and activates imager 16, with objective lens 72 moved to itsstart z position. The cross-sectional view of FIG. 4 illustrates anexample of the tissue sample 44 retained in carrier 49 upon tissuespecimen mounting platform 20 at a position during such movement fromimager 14 to imager 16. As shown in FIG. 12, with stage 18 having nowpositioned tissue sample 44 in view of objective lens 72 of microscopicimager 16, microscopic images 207 are captured by microscopic imager 16and shown by computer system 22 as video in a window 208 on screen 27 ofdisplay 23 from returned light received by such one of detectors 249 and253 providing reflectance microscopic images at 785 nm wavelength (asindicated by tab 211). The still macroscopic image 206 in window 200 hasoverlaid shaded box 209 showing the field of view of the microscopicimager 16 with respect to the tissue sample 44 for guiding or mapping tothe user the current location with respect to the surface of the tissuesample 44 facing substrate 42 where microscopic images are beingacquired. Other graphical elements, such as cross-hair lines 209 a arealso overlaid parallel with the x and y axes of the stage 18 on theselected still macroscopic image 206. Other graphical indicatorelement(s) of imager 16 position than box 209 may also be used. Thecurrent x, y and z positions 210 of the objective lens 72 are shown onthe screen 27, where depth along the z axis is calculated at orapproximate from surface 45 a into the tissue sample 44 from the startposition, and x and y position changes with movement of stage 18. Inthis manner, the macroscopic image in window 200 enables the user toguide the microscopic imager 16 to one or more selected locations forcapture of one or more microscopic images of the tissue sample 44. Eachmicroscopic image when captured is stored with location informationrelative to x and y coordinates in relation to the macroscopic image206.

Controls for microscopic imager 16 may be provided as graphical knobs212 selectable using mouse 29 to adjust depth by moving objective lens72 along its optical axis 73 towards or away from the substrate 42, orto turn on and off reflectance (785 nm) and fluorescent (488 nm)detection. Reflectance (785 nm) and fluorescent (488 nm) detection beingset in accordance with the particular filter or opening along filterwheels 248 and 252 in path of beam to their respective detectors 249 and253 when computer system 22 activates imager 16. The microscopic imagescaptured at such two different laser wavelength 488 nm and 785 nm areselectable in window 208 by using mouse 29 to select different ones oftabs 211 labelled by wavelength, while the microscopic imager 16 may beturned on or off by selecting button 213 with mouse 29. Buttons 201 maysimilarly be used to electronically zoom microscopic image 207 asdesired by the user. A still microscopic image of one of the microscopicimages 207 can be captured and stored in memory of computer system 22 byselecting a button 214, while different layouts of automatic imagecapture can be selected by one of three buttons 215. Such selection ofdifferent locations along a tissue sample 44 with automatic capture ofmicroscopic image(s) may be performed automatically by computer system22 sending signals to the x and/or y motors of stage 18 to enablemovement along x and/or y axes, and/or movement of objective lens 72along its optical axis 73. Captured microscopic images stored in memoryof computer system 22 using buttons 214 and 215 are displayed in avertical stack along window 216. Selection of a different x and ylocation along the tissue sample 44 may optionally be selected by theuser using joystick 30, keyboard 28, or by placement of a cursor andclicking using mouse 29 on a location on the tissue sample 44 shown instill macroscopic image 206 of window 200. When a different x and ylocation is selected, computer system 22 in response moves the stage's xand/or y motors accordingly. Further, the images in windows 200 and 208can be switched by the user as desired by the user clicking with mouse29 in the area within window 208. The microscopic images 207 displayedand/or stored in memory of computer system 22 can be used forpathological examination of tissue sample 44, such as performed in Mohsmicrographic surgery.

Other graphical user interfaces than shown in FIGS. 8, 10 and 12 may beprovided to display images captured by imagers 14 and 16. Where thecaptured images are associated with a patient or subject, a data entryscreen may be provided to start a session prior to start of capturingimages from imagers 14 and 16 so that patient or subject information mayentered and stored in memory of computer system in association withimages captured using buttons 202, 214, and 215. All or part of suchentered information may be displayed along window 217.

As described above, system 10 uses macroscopic images from macroscopicimager 14 for verification of the tissue sample orientation assuringthat the ex-vivo tissue sample 44 is properly oriented for imaging bymicroscopic imager 16, and if needed manipulated to a proper orientationagainst substrate 44 before the tissue sample is imaged by themicroscopic imager 16. Also, the still macroscopic image 206 captured isused as a guide or map image of the tissue sample 44 as the tissuesample is moved by stage 18 with respect to the microscopic imager 16 asindicated by graphical box 209.

From the foregoing description, it will be apparent that a system andmethod for macroscopic and microscopic imaging ex-vivo tissue has beenprovided. Variations and modifications in the herein described systemand method in accordance with the invention will undoubtedly suggestthemselves to those skilled in the art. Accordingly, the foregoingdescription should be taken as illustrative and not in a limiting sense.

1. A system for macroscopic and microscopic imaging ex-vivo tissuecomprising: a macroscopic imager; a microscopic imager; a stage formoving a substrate having optically transparent material supportingex-vivo tissue with respect to each of said macroscopic imager and saidmicroscopic imager to enable said macroscopic imager to capturemacroscopic images of said ex-vivo tissue when presented by said stageto said ex-vivo tissue, via said optically transparent material of saidsubstrate, and said microscopic imager to capture one or more opticallyformed sectional microscopic images on or within said ex-vivo tissue,when presented by said stage to said ex-vivo tissue, via said opticallytransparent material of said substrate, in which said macroscopic imagerand said microscopic imager each operate using a different detector inimaging said ex-vivo tissue; a computer system for controlling movementof said stage with respect to said macroscopic imager and saidmicroscopic imager, and receiving said macroscopic images and said oneor more microscopic images; a display for displaying said macroscopicimages and said one or more microscopic images when received by saidcomputer system; and a housing enclosing at least said macroscopicimager, said microscopic imager, and said stage, wherein said housingcomprises an opening for accessing said ex-vivo tissue upon saidsubstrate to permit manipulation of said ex-vivo tissue while saidmacroscopic images are viewable on said display with said substratepositioned by said stage to enable capture of said macroscopic images bysaid macroscopic imager, in order to assure said ex-vivo tissue isproperly positioned upon said substrate for subsequent capture of saidone or more microscopic images by said microscopic imager when presentedthereto by movement of said stage.
 2. The system according to claim 1wherein each of said macroscopic imager and said microscopic imager arein a different assembly fixed in position in said housing with respectto said stage prior to said ex-vivo tissue being presented to saidmacroscopic imager and said microscopic imager.
 3. The system accordingto claim 1 wherein said stage moves said substrate along x and yorthogonal axes, said macroscopic images and said one or moremicroscopic images are each two-dimensional images spatially alignedwith said x and y orthogonal axes, and optics of said macroscopic imagerfor imaging said ex-vivo tissue, and at least an objective lens ofoptics of said microscopic imager for imaging said ex-vivo tissue, eachhave an optical axis oriented to extend at least approximately parallelwith a z axis orthogonal to said x and y orthogonal axes.
 4. The systemaccording to claim 3 wherein said objective lens is movable along theoptical axis of the microscopic imager to adjust depth of said one ormore microscopic images within said ex-vivo tissue.
 5. The systemaccording to claim 1 wherein said macroscopic images of said ex-vivotissue are captured before capture of said one or more microscopicimages, and said macroscopic images of said ex-vivo tissue on saiddisplay enable verification that said ex-vivo tissue lies at leastsubstantially flush against a surface of said substrate by appearing infocus in said macroscopic images on said display, in which said openingin said housing permits manipulation to reposition at least one portionof said ex-vivo tissue upon said substrate when said at least oneportion appears unfocused in said macroscopic images on said displayuntil said at least one portion appears in focus in said macroscopicimages on said display. 6-7. (canceled)
 8. The system according to claim1 further comprising a member or another one of said substrate forapplying downward pressure onto said ex-vivo tissue against saidsubstrate when said one or more microscopic images are captured.
 9. Thesystem according to claim 1 wherein said housing has a cover movablebetween an open state and a closed down state, wherein said cover insaid closed state covers said opening of said housing to block ambientlight when at least said one or more microscopic images are captured bysaid microscopic imager, and said cover in said open state enablesaccess to said opening of said housing.
 10. The system according toclaim 1 wherein one of said macroscopic images is displayed on saiddisplay with display of said one or more microscopic images, and one ormore graphical elements are overlaid upon said one of said one or moremacroscopic images indicating at least a location of imaging by saidmicroscopic imager with respect to said ex-vivo tissue displayed in saidone of said one or more macroscopic images to guide in selection of oneor more locations along said ex-vivo tissue for imaging by saidmicroscopic imager.
 11. The system according to claim 1 wherein saidmacroscopic imager comprises optics which focus light from said ex-vivotissue at or near a surface of said substrate supporting said ex-vivotissue to said detector of said macroscopic imager, and light sourcesfor illuminating said ex-vivo tissue when said one or more macroscopicimages are captured by said macroscopic imager.
 12. (canceled)
 13. Thesystem according to claim 1 wherein said microscopic imager is operativeby confocal microscopy.
 14. The system according to claim 1 wherein saidcomputer system processes one or more of said macroscopic images todetermine presence of any unfocused portion of said ex-vivo tissue insaid one or more of said macroscopic images, and graphically indicatessaid any unfocused portion on said display of said macroscopic images,in which said opening in said housing permits manipulation to repositionsaid any unfocused portion of said ex-vivo tissue upon said substratewhere graphically indicated on said display until in focus in saidmacroscopic images on said display.
 15. The system according to claim 1wherein said microscopic imager is operated to capture one or more ofsaid one or more microscopic images at one or more locations along saidex-vivo tissue selected in one of said macroscopic images on saiddisplay.
 16. The system according to claim 1 wherein said opening insaid housing permits manipulation to reposition at least one portion ofsaid ex-vivo tissue upon said substrate when said at least one portionappears unfocused in said macroscopic images on said display until saidat least one portion appears in focus in said macroscopic images on saiddisplay.
 17. A method for macroscopic and microscopic imaging ex-vivotissue comprising the steps of: enclosing in a common housing at least amacroscopic imager, a microscopic imager, and a stage; placing ex-vivotissue on a substrate having optically transparent material; mountingsaid substrate on said stage, which is mounted in said housing to movesaid substrate with respect to each of said macroscopic imager and saidmicroscopic imager; moving said stage to present said ex-vivo tissue onsaid substrate to said macroscopic imager; capturing, with saidmacroscopic imager, macroscopic images via said optically transparentmaterial of said substrate; displaying said macroscopic images whencaptured with the aid of a computer system receiving said macroscopicimages; accessing said ex-vivo tissue upon said substrate via an openingalong said housing to permit manipulation for repositioning at least oneportion of said ex-vivo tissue upon said substrate when said at leastone portion appears unfocused in said macroscopic images on said displayuntil said at least one portion appears in focus in said macroscopicimages on said display while said step of displaying said macroscopicimages is being carried out; moving said stage to present said ex-vivotissue to said microscopic imager; capturing, with said microscopicimager, one or more optically formed sectional microscopic images on orwithin said ex-vivo tissue via said optically transparent material ofsaid substrate, wherein said step of capturing macroscopic images andsaid step of capturing one or more microscopic images are each carriedout using a different detector; and displaying at least one of saidmacroscopic images and said one or more microscopic images when capturedwith the aid of said computer system receiving said one or moremicroscopic images.
 18. The method according to claim 17 furthercomprising the step of verifying said ex-vivo tissue lies at leastsubstantially flush against a surface of said substrate by appearing infocus in said macroscopic images when displayed while said step ofdisplaying said macroscopic images is being carried out, and prior tosaid accessing step.
 19. (canceled)
 20. The method according to claim 18further comprising the step of placing a member or another one of saidsubstrate for applying downward pressure onto said ex-vivo tissueagainst said substrate after said steps of capturing macroscopic imagesand verifying are carried out, and before said step of capturing one ormore microscopic images.
 21. The method according to claim 17 furthercomprising the step of positioning a cover of said housing in an openstate when said accessing step in carried out, and in a closed state tocover said opening of said housing to block ambient light when said stepof capturing one or more microscopic images is carried out. 22.(canceled)
 23. An apparatus for imaging a non-histologically preparedtissue sample, said apparatus comprising: a macroscopic imager; amicroscopic imager for optically forming microscopic sectional imagesfrom scanned laser illumination at different selected depths in anon-histologically prepared tissue sample; a stage for moving saidnon-histologically prepared tissue sample, disposed upon one of twoopposing surfaces of a substrate, with respect to each of saidmacroscopic imager and said microscopic imager to enable saidmacroscopic imager to capture one or more macroscopic images of saidnon-histologically prepared tissue sample through said two opposingsurfaces of said substrate, and to enable said microscopic imager tocapture one or more of said microscopic sectional images of saidnon-histologically prepared tissue sample through said two opposingsurfaces of said substrate, when presented to said non-histologicallyprepared tissue sample by said stage, wherein said macroscopic imagerand said microscopic imager each operate using a different detector inimaging said non-histologically prepared tissue sample; a computersystem for controlling movement of said stage with respect to saidmacroscopic imager and said microscopic imager, and receiving said oneor more macroscopic images, and said one or more said microscopicsectional images; and a display for displaying said one or moremacroscopic images and said one or more said microscopic sectionalimages when received by said computer system.
 24. The apparatusaccording to claim 23 wherein said detector of said microscopic imagerrepresents one or more detectors each operated to enable capture of saidone or more optically formed microscopic sectional images.
 25. Theapparatus according to claim 23 wherein said detector of saidmacroscopic imager comprises an array of sensing elements enablingcapture of said one or more macroscopic images.
 26. The apparatusaccording to claim 23, wherein said non-histologically prepared tissuesample is separately imageable by each of said microscopic imager andsaid macroscopic imager as a result of movement of said stage.
 27. Thesystem according to claim 1 wherein said detector of said microscopicimager represents one or more detectors each operated to enable captureof said one or more optically formed sectional microscopic images. 28.The system according to claim 1 wherein said detector of saidmacroscopic imager comprises an array of sensing elements enablingcapture of said macroscopic images.